Abstract

Cultured ovarian granulosa cells (GCs) are essential models to study molecular mechanisms of gene reg-ulation during folliculogenesis. CCAAT enhancer binding proteins (CEBP) has been identified in theovary and is critical for follicular growth, ovulation and luteinization in mice. In the present study, hor-monal treatment indicated that luteinizing hormone (LH) and exogenous human chorionic gonadotropins(hCG) significantly increased the expression of CEBPin porcine GCs. By RNAi-Ready pSIREN-RetroQ-ZsGreen Vector mediated recombinant pshRNA vectors, CEBPgene was successfully knocked down inporcine GCs, confirmed by mRNA and protein level analyzed by real time PCR and western blot, respec-tively. We further found that knockdown of CEBPsignificantly increased the expression of p-ERK1/2.Furthermore, CEBPknockdown arrested the GCs at S phase of cell cycle, but had no effects on cell apopto-sis. More importantly, it markedly down regulated the concentration of estradiol (E2) and progesterone(P4) in the culture medium. To uncover the regulatory mechanism of CEBPknockdown on cell cycle andsteroids synthesis, we found that the mRNA expression of bcl-2 (anti-apoptosis), StAR and Runx2 (steroidhormone synthesis) was up-regulated, while genes related to apoptosis (Caspase-3 and p53), hormonalsynthesis (CYP11A1) and cell cycle (cyclinA1, cyclinB1, cyclinD1) were down-regulated, suggesting thatknockdown of CEBPmay inhibit apoptosis, regulate cell cycle and hormone secretions at the transcrip-tional level in porcine GCs. Furthermore, knockdown of CEBPsignificantly increased the expression ofPTGS2 and decreased the expression of IGFBP4, Has2 and PTGFR which are important for folliculogenesisin porcine GCs. In conclusion, this study reveals that CEBPis a key regulator of porcine GCs throughmodulation of cell cycle, apoptosis, steroid synthesis, and other regulators of folliculogenesis.

2. Materials and methods

2.6. ELISA for measurements of steroid hormonesAfter transfection with pshRNAi-2 and pshRNA-negative,respectively, the culture medium was collected at 48 h to mea-sure concentrations of E2 and Progesterone using the porcine ELISAkits. The sensitivity of E2 and Progesterone ELISA kit (Wuhan Col-orfulGene biological technology Co., Ltd, China) was 1.8–50 pmol/Land 100–2000 pmol/L, respectively. The ELISA procedure was per-formed according to manufacturer’s instructions.